Search

Microchimerism levels of recipient cells: frequency and impact on hematopoietic stem cell transplantation outcome

Major histocompatibility class I chain-related gene A (MICA) mismatching and development of anti-MICA antibodies after heart transplantation

Implementation of next generation sequencing (NGS) in routine clinical HLA laboratory typing brings numerous information but also challenges in data analysis. First 1268 samples, typed in our Tissue Typing Centre at University Hospital Centre Zagreb, with NGSgo-MX6-1 (GenDx) for HLA-A, B, C, DRB1, DQB1 and DPB1 loci revealed 19 novel HLA alleles. Among them, 9 (47.4%) were single amino acid substitution variants when compared to the most similar allele (HLA-A*01:200, A*02:836, A*02:1079, B*08:251, B*18:169, B*40:517, C*04:477, C*05:276 and DPB1*1146:01). Four alleles (HLA- B*15:18:08, B*18:37:02, B*27:02:06 and C*12:03:80) had silent substitution with no change in amino acid sequence and remaining six alleles (31.6%) showed only intronic mismatches to their most similar allele (HLA-A*03:01:01:112, A*11:01:01:44, A*11:01:01:83, B*07:02:01:107, B*08:01:01:67 and C*05:46:01:02). Most of the novel alleles were found at the HLA class I loci since the used tests cover the entire HLA-A, B and C genes. Class II loci are covered for exons 2 and 3 for HLA-DRB1 and DQB1, and exons 2-5 for HLA-DPB1. Besides these novel alleles, additional 102 alleles were observed only once. Looking at their status in the CIWD catalogue (version 3.0.0.), compiled from more than 8 million individuals, 46 alleles were common (≥1 in 10 000), 13 were intermediate (≥1 in 100 000), 16 were well-documented (≥5 occurrences) and 27 alleles were rare (not-CIWD). This diversity emphasizes the importance of continued research for identifying and publishing these alleles, not only to improve donor/recipient matching in hematopoietic stem cell transplantation program, but also to allow us to study in detail the HLA population diversity and mechanisms in which different demographic events and selective pressures have shaped HLA profiles of various populations.

The development of next-generation sequencing (NGS)for HLA typing has already had an impact on the scope and precision of HLA research. The distinctive allelic and haplotypic diversity found at the 4thfield reveals a high level of heterozygosity and specific haplotypic associations that were not apparent at the 2ndfield. In this study, allelic resolution HLA typing was obtained for 644 individuals from Croatia. The analysis was done for most common HLA-B~C and DRB1~DQB1 associations in order to examine whether specific haplotypes are formed depending on the 4thfield typing result. Allele HLA-B*08:01 revealed two predominant haplotypes: HLA-B*08:01:01:01~C*07:01:01:01 in 92 cases (100%) and B*08:01:01:02~C*07:02:01:01 in 6 cases (85.71%). For the allele HLA-B*44:02, three specific haplotypes are observed: HLA- B*44:02:01:01~C*05:01:01:01 (44/58 ; 75.86%), B*44:02:01:01~C*16:04:01:01 (14/58 ; 24.14%)and B*44:02:01:03~C*07:04:01:01 (5/5 ; 100%). Allele HLA-B*18:01:01:02 forms haplotypes with HLA-C*07:01:01:01 (45/76 ; 59.21%) and C*12:03:01:01 (25/76 ; 32.89%) while HLA- B*18:01:01:01 comes in a haplotype with C*05:01:01:01 (5/5 ; 100%). Alleles HLA-B*27:02 and B*27:05 most commonly form haplotypes with allele HLA-C*02:02, but this study showed that allele HLA-B*27:02:01:01 forms haplotype with HLA- C*02:02:02:03(15/15 ; 100%) while HLA-B*27:05:02:01 comes mostly with HLA-C*02:02:02:01 (39/54 ; 72.22%). For the HLA-DRB1~DQB1 associations the most remarkable example was seen for HLA- DRB1*13:02 which forms haplotype with either HLA- DQB1*06:04 or DQB1*06:09 but at 4thfield level haplotypes are HLA- DRB1*13:02:01:02~DQB1*06:04:01:01 (45/45 ; 100%) and HLA-DRB1*13:02:01:01~DQB1*06:09:01:01 (13/15 ; 86.67%). There-fore, this level of resolution obtained by NGS allows studying in detail the HLA diversity and may contribute as a useful reference source for future population studies, HLA-disease association studies and for improving donor recruitment strategies of bone marrow registries.

Immunization towards Human Leukocyte Antigens (HLA) can occur through pregnancy, blood transfusion, or previous transplant. Determination of unacceptable and acceptable HLA antigen mismatches is a critical decision step in finding a suitable donor for the HLA immunized kidney recipients. The Eurotransplant Acceptable Mismatch (AM) program enhance transplantation of highly sensitized patients by allocating the kidney from the donors with HLA antigens to which the patient has never formed the antibodies. Here, we present the characteristics of a cohort of 56 active and immunized patients (34 male and 22 female) on kidney transplant waiting list on 1st of July 2022. Among 56 immunized patients, 28 (50%) had a previous transplantation, 14 of them as the only immunizing event, 11 received transfusion as well, while 3 female patients also had a pregnancy as the third immunizing event. A total of 30 (54%) immunized patients have the immunization to both class I and class II HLA antigens, 19 (34%) patients have only HLA class I immunization and 7 (12 %) are immunized only to class II HLA antigens. In the terms of virtual panel reactive antibodies (vPRA), 15 patients have vPRA over 98%, which makes them eligible for AM program. Among them, 14 patients (93%) had previous transplantation as the main immunizing event. Three of these patients are already registered in AM program, while five more are in a process of the registration. All together the HLA immunization profile of our patients is pointing to the previous transplantation as a major event leading to the high immunization that makes the patient eligible for the AM program. This also emphasizes the importance of donor-recipient HLA matching in the first transplantation, especially for children and young patients in which second transplantation is expected in the future.

Investigation of allele and haplotype distribution of genes pertaining to Human Leukocyte Antigen complex (HLA) is relevant for population studies but also in hematopoietic stem cell transplantation (HSCT) from HLA matched unrelated donor. This study encompassed 10 000 healthy unrelated volunteer donors from Croatian Bone Marrow Donor Registry (CBMDR). All subjects were analyzed for HLA-A, -B, -C and -DRB1 loci by one of the PCR based methods. A total of 49 different alleles on HLA-A locus was observed, 92 alleles on HLA-B locus, 36 alleles on HLA-C locus and 56 alleles on HLA-DRB1 locus. Most frequent allele for HLA-A locus was A*02:01 (29, 1%), for HLA-B locus most frequent was B*51:01 (10, 5%), HLA- C*07:01 (15, 8%) was the most frequent allele on HLA-C locus while for HLA-DRB1 most frequently observed allele was DRB1*03:01 (10, 6%). Most common haplotype was HLA- A*01:01C*07:01B*08:01DRB1*03:01 (4, 7%). Comparison of allele and haplotype frequencies with other populations of European anchesty showed some specificities of our population. Obtained results emphasize importance of existence of small and medium size registries of HSC donors due to their contribution to HLA allele and haplotype diversity identification. Finally, data provided by this study will serve to improve the procedure of finding the best donor for a given patient in HSCT program.

Introduction Rituximab is a genetically engineered recombinant chimeric monoclonal antibody directed against CD20 antigen expressed on the surface of B cells. It can activate the complement-mediated cytotoxicity and in that way reduce B cells in the peripheral blood. It is used as a treatment of several autoimmune disorders and B-cell malignancies but also to control and prevent humoral rejection in solid organ transplantation (SOT). However, rituximab when present in the sera of patients awaiting SOT causes false positive crossmatch (CM) due to the structural homology of Fc receptors on activated T-cells. This could be a possible explanation of the same and provide insight into a novel mechanism of action of rituximab. Case report A 49-year-old male patient with ANCA-vasculitis and chronic kidney insufficiency was assessed for kidney transplantation from a family member in 2018 but was not transplanted due to the clinical condition of the potential donor. Luminex Single Antigen Beads (SAB) testing was performed and turned to be positive for HLA-B73 with virtual panel reactive antibodies (vPRA) 1%. The patient was registered on Eurotransplant waiting list in April 2021 and got his first kidney offer in April 2022 but the acceptance had to be withdrawn due to the unexpected positive CM result. Positive CM was unexplainable by the previous HLA and non-HLA antibody detection and identification tests. Clinical data revealed that patient started rituximab therapy in April 2019 and received total of five doses, the last one in November 2021. In the period from February to June 2022 patient had four more unexplainable positive CMs. The CM tests were performed with serum samples drawn in December 2021 and March 2022, i.e. one and four months after the rituximab therapy. In July 2022, patient received a cadaveric kidney offer, Luminex SAB testing confirmed the absence of DSAs and negative CM was obtained with serum samples drawn in June 2022 and at the day of transplantation i.e. with samples drawn 8 months after the last rituximab dose. Conclusion In the light of the growing use of rituximab in various medical conditions, it is vital to know the clinical and drug history of the recipient before interpretation of any immunological test. The SAB assay is a valuable technique in this setting as the presence of rituximab does not influence it, while CM results are dependent on the time frame of the last rituximab dose.

In an effort to avoid alloreactive responses afterhaploidentical hematopoietic stem cell transplantation(haplo-HSCT) it is important to identify and minimizethe number of HLA-mismatches between donor andrecipient. One of the possibilities is to apply algorithmsthat quantifies the structural and functional differencesbetween a recipient's and donor's HLA antigens. We ana-lyzed a cohort of 64 adult patients with hematologicmalignancies who received a transplant from a relatedhaploidentical donor at the Clinical Hospital CenterZagreb. HLA eplet- mismatches (EM) calculated by theHLAMatchmaker algorithm as well as PIRCHE score(PS) data among patients and their haplo-identicaldonors were analyzed in both directions, graft versus host(GvH) and host versus graft (HvG). The effect on graftversus host disease (GvHD) and relapse incidence wereestimated. The median of total HLA class I and class IIEM was 38 (range: 11–60), with the mismatches predomi-nately found in the HvG direction (median 23, range: 5–52) as compared to the GvH direction (median 15, range:0–25). The median HLA class I PS in the GvH directionwas 16 (range: 1–48), and the median HLA class I PS inthe HvG direction was 16 (range: 0–59). Patients withHLA class I+II EM 15 (26.3%) demon-strated significantly higher incidence of relapse(P=0.0243 ; RR=2.80 ; 95% CI [1.14– 6.86]). The patientswith HLA class I PS >20 in the GvH direction had a sig-nificantly higher (P=0.0081) incidence of GvHD whichwas also confirmed in a multivariate analysis (P=0.0420 ; OR=4.09 ; 95% CI [1.05–15.89]). The resultsof this study confirm the HLAMatchmaker and PIRCHEscoring algorithms as a useful tool in prediction of per-missible HLA mismatches and improve the selection of abest haplo-donor.

Introduction Antibodies directed against donor mismatched Human Leukocyte Antigens (HLA) can cause graft loss after transplantation. Many studies describe the influence of donor-recipient HLA class I mismatches (MM) on transplant outcome, but effects of HLA class II MMs are still unknown, especially for HLA-DP locus. Some studies show that there are no deleterious effects of HLA-DP mismatching in first transplants, but that there is a significant impact on re-transplants. As avoiding such MMs is crucial for highly sensitized patients, the Eurotransplant Acceptable mismatch (AM) program has been initiated and is based on finding a donor with HLA antigens to which the patient has never made antibodies. Still, long term analysis revealed that most highly sensitized patients were most likely to die or be removed from the list than transplanted. This led to the development of clinical trials for desensitization therapies that would enable HLA-incompatible transplantation. European Medicines Agency has recently approved novel agent, the IgG-degrading enzyme (Imlifidase) that cleaves all four human subclasses of IgG and enables rapid degeneration of anti-HLA donor specific antibodies after drug administration. Case Study Here, we report a case of a 39-year-old female patient who received a first kidney transplant in 1990 from a deceased donor and suffered from graft rejection 12 years later. In January 2015 the patient received a second deceased donor transplant, but this graft failed right after the transplantation. Being a highly sensitized patient, with two previous cadaveric transplantations, pregnancy and transfusion, there were no suitable donor offers for her afterwards. All available living donors were excluded due to positive DSAs and strong positive crossmatch (CM), among which CM with father stood out as weak positive, probably due to lower titer of HLA class I DSAs. For all these reasons she applied for the AM program in which she was registered in November 2021. Since then, she got two kidney offers with no HLA-A, B, C, DR and DQ MMs, but both offers were rejected due to the poor organ quality. It still remains the question whether they would be suitable offers since there was no information on HLA-DP typing of the donors. The main challenge for this patient is high HLA-DP sensitization and whether she should be transplanted across HLA-DP DSAs and possibly a positive crossmatch. Conclusion Here we present a highly sensitized patient with a high HLA-DP sensitization that could be a candidate for Imlifidase administration in order to expand the pool of possible donors.

The ability of the mother’s immune system to “tolerate” the embryo which presents “non-self” Human Lekocyte Antigens (HLA) from the father is essential to achieve a successful pregnancy. This immunotolerance is achieved through interactions between the killer-cell immunoglobuline like receptors (KIR) on maternal uterine NK cells and fetal HLA-C antigens. Recent studies have revealed an increased risk of pregnancy complications/failure in mothers with the KIR AA haplotype when the fetus has more HLA-C2 epitope group genes than the mother. So the KIR and HLA-C genotyping of the mother and HLA-C genotyping of the father can help predict compatibility between maternal uterine KIRs and potential HLA-C presented by the embryo. In the Tissue typing Centre Zagreb so far we performed KIR and HLA-C genotyping and analyze the combinations of KIR and HLA-C genes in three couples (mother and partner) referred from a gynecological clinic due to reproductive infertility. All individuals were tested for HLA-C and KIR poylmorphisms using PCR- SSP method. None of the couples were deleterious KIR AA haplotype/ HLA-C2 epitope mother/partner combination. In conslusion, genetic testing for KIR/HLA-C could be additional diagnostic tool and should be implemented in algorithm of procedures for diagnosing infertility and help to direct medical interventions in order to achieve a successful pregnancy.

Importance of HLA-DPB1 and DPA1 genes in solid organ and hematopoietic stem cell transplantation outcome has been increasingly studied in recent years and therefore the aim of this study was to analyze HLA-DBP1 and DPA1 allele and haplotype frequencies in the Croatian population (N=124). Eighteen different HLA-DPB1 alleles were observed among tested subjects, with HLA-DPB1*04:01 allele being the most frequent one (36.7%), followed by DPB1*04:02 and DPB1*02:01 alleles (16.1% and 15.7%, respectively). HLA-DPA1 locus showed limited diversity with only 6 different alleles, among which the most common was HLA-DPA1*01:03 allele (76.2%). Despite such small cohort and low HLA-DPA1 polymorphism one very rare allele was detected (DPA1*01:06:02). Percentage of heterozygotes was 79.8% for HLA-DPB1 and only 37.9% for HLA-DPA1 locus. Haplotype analysis revealed 24 different haplotypes, with HLA- DPB1*04:01~DPA1*01:03 and HLA- DPB1*04:02~DPA1*01:03 haplotypes being most commonly observed (36.3% and 16.1%, respectively). This study clearly demonstrates the necessity to further investigate HLA-DPB1 and DPA1 loci since a very limited number of studies focused on these genes and especially the perception of HLA-DPA1 as low polymorphism locus might change with more data available at higher resolution level.

GOALS: Pretransplant donor-specific antibodies (pre-tx DSA) are associated with an increased risk of antibody-mediated rejection (AMR) and allograft loss. Following transplantation (tx), the level of DSAs may increase, decrease, remain persistent or be completely cleared from the recipient's blood and thus should be carefully and continuously monitored in the post-transplant period. MATERIAL AND METHODS The patient was a 46-year-old male waitlisted in September 2019 for the second kidney tx. The presence of HLA antibodies detected with the Luminex Single Antigens beads method (LSA1, LSA2) for HLA class I and class II antibodies showed that the patient was highly immunized, with a highest virtual panel of reactive antibodies (vPRA) of 92%. The complement-dependent cytotoxicity (CDC) PRA at the time of tx was 44% pointing to the presence of cytotoxic complement binding HLA class I antibodies. RESULTS In January 2020, the patient received kidney offer from cadaveric donor. The donor/patient ABCDRDQ mismatch was 21011, with Luminex results positive for the presence of pre-tx HLA class I DSAs with low median fluorescence intensity (MFI: 1300- 1700). The CDC crossmatch was negative, excluding the presence of cytotoxic HLA class I DSAs. The plasmapheresis was performed and Luminex test results turned negative for the presence of pre-tx DSAs. The tx was carried out and the patient continued to be carefully immunologically monitored. Seven months after tx, the pre-tx existing DSAs reappeared with the same low MFI values as before tx and persisted equally positive till today. The kidney function is stable, without the episodes of AMR. CONCLUSION The decision about performing tx with pre-tx DSA present should be made carefully and detailed pre- tx and especially post-tx DSA monitoring are important to improve individual risk stratification for kidney allograft loss.

INTRODUCTION: Detection of human leukocyte antigen (HLA) donor-specific antibodies (DSAs) is important for evaluating humoral immune status of patients pre- and post-transplantation. Both recipient and donor HLA allele typing data, ideally for all 11 immunogenic HLA loci (HLA-A, - B, -C, -DRB1, DRB3/4/5, -DQA1/DQB1 and - DPA1/DPB1), are the prerequisite for the accurate identification and detection of DSAs. In a majority of patients that have been transplanted long ago or transplanted outside our transplantation program the main obstacle for this analysis is the lack of stored donors’ blood or tissue samples. CASE REPORT: Here we report the methodology, which we used to overcome the situation of the missing donors’ blood samples by using the tissue undertaken from the kidney allograft biopsy. In three cases kidney transplantations (Ktx) were performed 19, 18 and 13 years ago and in one case Ktx was performed abroad. A tiny sample of kidney allograft tissue was used for DNA extraction and further HLA typing with polymerase chain reaction- specific sequence primers (PCR-SSP) and/or polymerase chain reaction-sequence-specific oligonucleotides (PCR-SSO) methods. In all four cases the obtained HLA typing result represented a “mixture” of both patient and donor HLA phenotypes, which allowed us to determine only the mismatched donor HLA antigens by deduction and comparison with the patients’ own HLA phenotype. In three cases, DSAs were positive: in case #1 for DRB4 and DQB1 alleles ; in case #3 for DPB1 and in case #4 for DQA1 alleles. In case #2, the locus specificity in obs. was DRB3 and the presence of DSAs was excluded. CONCLUSION: In summary, in all cases the kidney allograft biopsy sample was a useful source for DNA extraction and further HLA typing of the sample, which enabled us to identify possible donor immunogenes to which patient could form DSAs and to analyse the patients’ antibody profile for the presence or absence of DSAs.

Relapse represents a frequent and severe complication after HSCT. In most cases relapse has recipient's origin and leads to status of mixed chimerism. However, in some cases it may be due to specific immune escape by leukemia via genomic loss of mismatched HLA haplotype. Aim of this study was to retrospectively analyze incidence of HLA loss in sixty patients transplanted with haploidentical donor at UHC Zagreb (2012-2019). Post HSCT chimerism status was monitored by qPCR. Mixed chimerism monitoring results identified 22 patients at higher risk for relapse, which was clinically confirmed for 12 (54.5%) of them. All 12 patients were tested for HLA loss using HLA typing by PCR-SSP. Patients were also tested by qPCR for 10 markers targeting some of the most frequent alleles of HLA-A, -C, and -DPB1 loci. HLA loss was detected in two patients. Patient 1 relapsed 6 years after HSCT, at which time chimerism monitoring detected 80% of patient cells. HLA typing revealed loss of patient’s mismatched allele at informative loci HLA-A, -B, - C. Patient 2 relapsed 5 months after HSCT, with chimerism monitoring result of 2% of patient cells. Loss of patient’s mismatched haplotype was detected at loci HLA-A and -DPB1. Ten patients relapsed without HLA loss with mixed chimerism monitoring results. In all cases PCR-SSP testing was successful in detecting mismatched patient alleles. The results suggests that PCR-SSP method is sufficiently sensitive and useful for detection of patient-specific HLA haplotype loss, providing clinically relevant information for relapse treatment even in cases with low level of patient cells in peripheral blood.

Chimerism analysis provides valuable information about the efficacy of the hematopoietic stem cell (HSC) transplantation as treatment for various hematologic diseases and disorders. The method of choice for this analysis has recently shifted form short tandem repeat (STR) loci detection towards real-time PCR based methods due to their higher sensitivity. The present study was undertaken in order to evaluate whether the STR analysis should remain a preferred choice in cases when a patient receives a second HSC transplant from a different donor. The study included 17 patients for whom the first transplantation did not achieve favourable outcome, so a second transplantation, with a different donor, was performed. Samples of patient before HSCT, both donors and patient after second HSCT were amplified for 21 STR markers using a commercial kit (Aneufast™ QF-PCR Kit, Genomed Biotech, Harrow, UK). Only the second donor was present in 16 cases, while in the remaining one case, both the first and the second donor were detected in a 36% vs. 64% ratio in the post HSCT sample. This case supports the use of STR markers in chimerism evaluation in all cases when there is a possibility that more than two distinct genotypes are present in tested sample as is the case with second HSCT. As opposed to biallelic insertion/deletion polymorphisms analysed in real- time PCR based protocols, STR markers provide sufficient polymorphism to reliably distinguish all genotypes present in post HSCT sample.

Hematopoietic stem cell donor registries implement different strategies for improvement of their work, mainly focusing on increasing resolution of HLA typing and number of tested HLA loci on one hand, and young male donor recruitment on the other. The aim of this analysis was to assess the Croatian Bone Marrow Donor Registry (CBMDR) strategy, from the HLA diversity point of view, of age and gender based donor inclusion in the set appropriated to HLA typing by a nextgeneration sequencing method. The CBMDR currently lists >56, 000 donors, with prevalence of female (F) donors both in the total number of CBMDR donors (N(F)=33, 963 ; 60.3%) as well as in different age groups: 18-30yrs (F-62.2%) ; 31-50 yrs (F-60.1%) ; >50yrs (F- 57.7%). HLA phenotypes at HLA-A, -B, -DRB1 loci split level (excluding HLA-B*14, -B*15 and -B*40) were analyzed. The number of different HLA phenotypes discovered in the total number of CBMDR donors is 36, 270 ; which represents 0.27% of all possible combinations. Two HLA phenotypes were seen in >100 donors, 235 HLA phenotypes were observed frequently (11-100x) and 8, 237 HLA phenotypes were detected more sporadically (2-10x). The remaining 27, 796 different HLA phenotypes appeared only once, representing a group of unique HLA phenotypes. The distribution of unique HLA phenotypes in different age groups did not provide evidence of one group being more heterogeneous, since approx. 50% of donors in each group carried a unique HLA phenotype. Similar results were obtained when analysis was extended on female and male donors within the same age group. Finally, analysis of the number of donors carrying unique HLA phenotypes originating from different regions of Croatia also revealed no significant differences. To conclude, based solely on HLA phenotype diversity perspective, each donor group, regardless of the age, gender and origin, contributes equally to HLA phenotype variety of CBMDR, and should be included in future highresolution HLA typing.

Haploidentical hematopoietic stem cell transplantation (HSCT) outcome depends highly on selection of a best donor which can be improved by including additional factors such as killer immunoglobulin-like receptors (KIR) into consideration. This retrospective study involved 52 patients (P) and their donors (D) in the haplo-HSCT program in UHC Zagreb who were analyzed for different models of KIR impact on HSCT outcome. Effects of KIR ligand-ligand mismatch (MM), KIR inhibitory receptor-ligand MM in GvH direction, KIR AA/Bx haplotype, number of activating KIR (aKIR) genes and presence of specific aKIR gene in the donor on overall survival (OS) and relapse incidence were estimated. No difference in KIR gene distribution between P and D was observed but three aKIR (2DS1, 2DS5, 3DS1) genes had significantly higher frequency among P (P=0.039 ; P=0.005 ; P=0.010) and D (P=0.010 ; P=0.048 ; P=0.010) in comparison to their frequencies in the Croatian population. Lower occurrence of KIR haplotype AA and higher occurrence of haplotype Bx was revealed among P (AA- 25.0% ; Bx-75.0%) and D (AA-28.8% ; Bx-71.2%) when compared to controls (AA-33.6% ; Bx-66.4%). Fifteen P-D pairs (28.8%) shared identical KIR genotypes. In the remaining 37 pairs (71.2%) that differed in KIR genotype combinations, the P-D KIR haplotype combination with highest frequency was Bx(P)/Bx(D) (59.6%) while all other combinations were similarly represented (AA(P)/AA(D)- 13.5% ; AA(P)/Bx(D)-11.5% ; Bx(P)/AA(D)-15.4%). All P-D pairs had at least 1 KIR ligand MM with the highest percent of those with 2 MM (38.5%). The OS rate for the whole cohort was 57.7% with 30.8% of relapse incidence (median follow-up time of 355 days). None of investigated variables had significant impact on OS or relapse incidence in univariate and multivariate analyses. Obtained results might be in part explained by small sample size, so further evaluation on larger transplant cohort is required to define the KIR impact on haplo HSCT outcome.

Haploidentical hematopoietic stem cell transplantation (HSCT) can extend opportunity for transplantation for almost all patients who lack an HLA-matched donor. Genomic loss of mismatched HLA haplotype (HLA loss) is a clinically-relevant mechanism of leukemia immune evasion and relapse after haploidentical HSCT. The aim of this study was to retrospectively analyze incidence of HLA loss in sixty patients transplanted with haploidentical donor at UHC Zagreb in period 2012-2019 for treatment of acute leukemia (N=28), lymphoma (N=26) or other hematological malignancies (N=6). Post- HSCT chimerism status was monitored by Real-Time PCR (qPCR). Mixed chimerism monitoring results identified 22 patients at higher risk for relapse, which was clinically confirmed for 12 (54.5%) of them. All 12 patients were tested for HLA loss at all HLA class I and class II informative loci using HLA typing by PCR-SSP. HLA loss was detected in two patients. Patient 1 relapsed 6 years after HSCT, at which time chimerism monitoring detected 80% of patient cells. HLA typing revealed loss of patient’s mismatched allele at all three informative loci (HLA-A, - B, -C). Patient 2 relapsed 5 months after HSCT, with chimerism monitoring result of 2% of patient cells. In this patient HLA-A and -DPB1 loci were informative and at both mismatched allele loss was detected. Ten patients relapsed without HLA loss with mixed chimerism monitoring results and the level of patient cells ranging from 2%-80%. In all cases PCR-SSP testing was successful in detecting mismatched patient alleles, however in cases of lower level of patients’ cells (2-15%) bands were less intensive, but still clearly positive. The results suggest that the PCR-SSP method is sufficiently sensitive and can be used for detection of patient-specific HLA haplotype loss, providing clinically relevant information for relapse treatment even in cases with low level of patient cells in peripheral blood.

Istraživanja raspodjele alela i haplotipova glavnog sustava tkivne snošljivosti (engl. Human Leukocyte Antigen, HLA) provode se u svrhu populacijskih istraživanja kao i u programu transplantacije krvotvornih matičnih stanica (TKMS) od HLA podudarnog nesrodnog davatelja. Ovim radom obuhvaćena je skupina od 10 000 zdravih nesrodnih dobrovoljnih davatelja iz Hrvatskog registra dobrovoljnih davatelja krvotvornih matičnih stanica (HRDDKMS) te skupina od 261 nesrodnog bolesnika u programu nesrodne TKMS. Svim ispitanicima analizirani su geni HLA-A, -B, -C i -DRB1 jednom od molekularnih metoda. Obradom podataka o učestalosti alela i haplotipova HLA dobivene su vrijedne informacije o raznolikosti alela HLA te čestim, rijetkim i vrlo rijetkim alelima i haplotipovima HLA u Hrvatskoj. Rezultati rada ističu važnost održavanja manjih i srednjih registara dobrovoljnih davatelja KMS čime se doprinosi prepoznavanju raznolikosti alela i haplotipova HLA. Također, ističu ulogu HLA kao ograničavajućeg čimbenika u pronalaženju fenotipski HLA podudarnog nesrodnog davatelja te služe unaprjeđenju postupka pronalaska najboljeg mogućeg davatelja za pojedinog bolesnika. Investigation of allele and haplotype distribution of genes pertaining to Human Leukocyte Antigen complex (HLA) is relevant for population studies but also in hematopoietic stem cell transplantation (HSCT) from HLA matched unrelated donor. This study encompassed 10 000 healthy unrelated volunteer donors from Croatian Bone Marrow Donor Registry (CBMDR) and 261 patients who underwent unrelated HSCT. All subjects were analyzed for HLA-A, -B, -C and -DRB1 genes by one of DNA analysis based methods. HLA allele and haplotype frequency analysis provided valuable information about HLA allelic polymorphism as well as data about common, rare and very rare HLA alleles and haplotypes in Croatia. Obtained results emphasize importance of maintaining small and medium size registries of HSC donors due to their contribution to HLA allele and haplotype diversity identification. Results also highlight the role of HLA as a limitation factor in identification of HLA phenotypically matched unrelated donor. Finally, data provided by this study will serve to improve the procedure of finding the best donor for a given patient.

Istraživanja raspodjele alela i haplotipova glavnog sustava tkivne snošljivosti (engl. Human Leukocyte Antigen, HLA) provode se u svrhu populacijskih istraživanja kao i u programu transplantacije krvotvornih matičnih stanica (TKMS) od HLA podudarnog nesrodnog davatelja. Ovim radom obuhvaćena je skupina od 10 000 zdravih nesrodnih dobrovoljnih davatelja iz Hrvatskog registra dobrovoljnih davatelja krvotvornih matičnih stanica (HRDDKMS) te skupina od 261 nesrodnog bolesnika u programu nesrodne TKMS. Svim ispitanicima analizirani su geni HLA-A, -B, -C i - DRB1 jednom od molekularnih metoda. Obradom podataka o učestalosti alela i haplotipova HLA dobivene su vrijedne informacije o raznolikosti alela HLA te čestim, rijetkim i vrlo rijetkim alelima i haplotipovima HLA u Hrvatskoj. Rezultati rada ističu važnost održavanja manjih i srednjih registara dobrovoljnih davatelja KMS čime se doprinosi prepoznavanju raznolikosti alela i haplotipova HLA. Također, ističu ulogu HLA kao ograničavajućeg čimbenika u pronalaženju fenotipski HLA podudarnog nesrodnog davatelja te služe unaprjeđenju postupka pronalaska najboljeg mogućeg davatelja za pojedinog bolesnika.

Information on the precise content of HLA haplotypes is needed for transplantation, disease association, anthropological and epidemiological studies. In this study we aimed to analyse HLA- DRB1*04~DQB1 haplotype diversity at a high- resolution level in DRB1*04-positive individuals (N=734). The high-resolution level typing of HLA- DRB1 and DQB1 loci was performed using the Polymerase Chain Reaction - Sequence Specific Primers (PCR-SSP) method. A total of 10 different DRB1*04 alleles (DRB1*04:01- 08, DRB1*04:15 and DRB1*04:50) and nine different DQB1 alleles (DQB1*03:01-05, DQB1*03:07, DQB1*02:02, *04:01-02) have been determined, composing 29 different haplotypes. The most common DRB1*04 alleles were DRB1*04:01 (30.6%), DRB1*04.02 (25.2%), DRB1*04:04 (19.7%) and DRB1*04:03 (13.5%). The most prevalent DQB1 allele was DQB1*03:02 (85%), followed by DQB1*03:01 (7.5%). The two most frequent haplotypes were DRB1*04:01~DQB1*03:02 and DRB1*04:02~ DQB1*03:02 (35.2% and 27.4%, respectively). The haplotype analysis performed for each respective HLA-DRB1*04 allele revealed the most prevalent association with DQB1*03:02 for most of them. The most striking one was exclusive association of DRB1*04:02 with DQB1*03:02, as it was observed in all DRB1*04:02 positive cases (N=201). On the other side, DRB1*04:07 was most commonly found with DQB1*03:01 (83% of DRB1*04:07 positive cases) while DRB1*04:08 was most commonly found with DQB1*03:04 (82% of DRB1*04:08 positive cases). The analysis also showed DRB1*04:03~DQB1*03:05 as an exclusive haplotype association, as it was found on all DQB1*03:05 positive haplotypes (N=9). DRB1*04:05 was the most frequent allele found in an unusual association with DQB1*02:02 (85 % of DQB1*02:02 positive haplotypes). These results provide information on HLA-DRB1*04~ DQB1 haplotype diversity in the Croatian population that can be implemented in the strategic planning in unrelated hematopoietic stem cell transplantation increasing the likelihood of finding a suitable donor.

Population studies focused on HLA genes, due to their high allelic diversity, are of great importance for tracking changes in evolutionary history of different populations, identification of conserved combinations of different alleles and linkage disequilibrium among them. The aim of this study was to compare the distribution of three most common HLA 344 ABSTRACTS haplotypes in the general Croatian population with their regional distribution in a total of 4792 volunteer unrelated donors from ten different recruitment cities across Croatia (N=243 to N=1071). The capital, Zagreb, was excluded from regional distribution analysis. All individuals were tested for HLA-A, -B, -C, and -DRB1 alleles by PCR-SSO and PCR-SSP methods. The previously published results were used for general population haplotype frequencies. The most common haplotype in the general population, as well as in all ten cities was HLA-A*01:01~B*08:01~ C*07:01~DRB1*03:01. Its regional frequency distribution ranged from 3.7% to 7.0%, and did not show differences in regional distribution nor when compared with general population (4.2%). HLA- A*02:01~B*18:01~C*07:01~DRB1* 11:04, as the second ranked haplotype in general population (1.5%), showed regional distribution frequency differences ranging from 0.8% to 3.2%, with a haplotype position ranking from 2 to 16. The observed differences did not reach statistical significance. The third most common haplotype in general population HLA-A*03:01~B*07:02~C*07:02~ DRB1*15:01 (1.2%) showed high statistically significant regional distribution differences, with a frequency range from 0.2% to 3.0%. It was ranked as second or third most common haplotype in a total of four cities, in four cities it had haplotype position ranking from 5-10, in one city it was present only once while in one city it was not detected at all. This type of study would be useful for optimizing donor recruitment strategies and registry planning.

In 2007 Croatia became a member of Eurotransplant and since then participates in allocation program in which the kidney recipient's selection list was based on a scoring system which includes a number of HLA-A, - B and -DR mismatches (MM) between patient and cadaveric donor. The aim of this study was to compare HLA-A, -B, -DR antigen distribution among cadaveric donors from Croatia (CRO donors) and donors from other Eurotransplant countries (ET donors) ; to determine the number of MM within patients who received the kidney from the CRO or ET donor and to estimate the benefit of organ exchange for our patients on the kidney waiting list. The study included 861 patients and 707 cadaveric donors (CRO donors N=422, ET donors N=285). Statistically significant differences of specific HLA-A, -B and -DR antigen frequencies between CRO and ET donors were observed. HLA antigens significantly more present among ET donors were HLA-A29 (p=0.0161), B7 (P=0.0048), B8 (P=0.0316), B40 (B60) (P=0.0404) and B44 (P =0.0061) while B35 (P=0.0022), DR11 P=0.0350) and DR16 (P=0.0001) were more frequently present in the CRO donor group. Consequently, patients positive for HLAB7, B8, or B44 antigens statistically significantly more often received a kidney from ET donors (P=0.0016, P=0.0198 and P=0.0312, respectively), while HLA-DR16 positive patients received a kidney more frequently from the CRO donors (P =0.0306). The ABDR MM 000 was more frequently present in the case of transplantation from ET donor (P=0.0005), while MM 222 was significantly more frequent when the donor was from Croatia (P=0.0277). Differences in HLA antigen distribution among populations of Eurotransplant member countries are reflected to kidney allocation and patients from Croatia. Overall MM analysis shows 302 ABSTRACTS that a larger pool of organ donors allows higher probability of HLA highly matched transplantation.

Aim: Endemic nephropathy (EN) is classified as a tubulointerstitial progressive chronic disease that affects kidneys with a slow clinical course. Disease occurs in rural communities along the river flows of the Danube river basin in Croatia, Bosnia and Herzegovina, Serbia, Bulgaria and Romania. In Croatia, the endemic focal point includes fourteen villages and affects population of about 10 000 people located in the BrodskoPosavska County on the Sava River bank, west of Slavonski Brod. Over the past period, the influence of hereditary and environmental factors on the appearance of EN (heavy metals, microelements, various viruses and bacteria, soil, drinking water, polycyclic hydrocarbons aromatic compounds) were investigated. Recently, next generation sequencing nominated three genes tightly connected to process of angiogenesis as candidate genes for predisposition to Balkan endemic nephropathy, one of them (KCNK5) being located on chromosome 6p21.2, in close proximity to the HLA region. Methods: Prompted by this finding, we investigated HLA-A, -B, and -DRB1 alleles and haplotypes in the population of patients with EN (N=103) and matched healthy controls (N=190). All individuals were tested by PCR-SSO for low resolution typing and PCR-SSP to obtain a high-resolution typing. Results: The results showed higher presence of HLA-DRB1*04:02 allele in EN (P=0.013, OR=3.006, 95% CI=1.28- 7.07), in contrast to the lower frequency of HLA- A*01:01, B*57:01 and B*27:05 alleles (P=0.002, OR=0.373, 95% CI=0.19-0.72 ; P=0.032, OR=0.339, 95% CI=0.13-0.90 and P=0.006, OR=0.098, 95% CI=0.01-0.74, respectively). Moreover, when EN patient’s HLA haplotypes were compared to controls, two haplotypes were present with higher frequency within EN patients group, HLA- A*02:01~B*27:02~DRB1*16:01 and HLA- A*26:01~B*38:01~DRB1*04:02 (P=0.002, OR=0 and P=0.054, OR=4.702, 95% CI=0.90- 24.45, respectively) while HLA-A*02:01~B*57:01~DRB1*16:01 (P=0.031, OR=0) haplotype showed a significantly lower frequency. Two other haplotypes, HLA- A*02:01~B*27:05~DRB1*01:01 and HLAA*01:01~B*57:01~DRB1*07:01, were also less frequent among EN patients, but this difference did not reach statistical significance. Conclusion: The results point toward genetic susceptibility to EN indicating the necessity of further studies.

Endemic nephropathy (EN) is a chronic kidney disease affecting populations in several countries, with an endemic focus in eastern Croatia with a population of about 10, 000 people. EN is a multifactorial disease with polygenic predis position to environmental risk agents. Recently, next generation sequencing nominated three genes tightly connected to process of angiogenesis as candidate genes for predispos tion to Balkan endemic nephropathy, one of them (KCNK5) being located on chromosome 6p21.2, in close proximity to the HLA region. Prompted by this finding, we investigated HLA-A, - B, and -DRB1 alleles and haplotypes in the popu lation of patients with EN (N=103) and matched healthy controls (N=190). All individuals were tested by PCR-SSO for low resolution typing and PCR-SSP to obtain a high- resolution typing. The results showed higher presence of HLA-DRB1*04:02 allele in EN (P=0.013, OR=3.006, 95% CI=1.28-7.07), in contrast to the lower frequency of HLA- A*01:01, B*57:01 and B*27:05 alleles (P=0.002, OR=0.373, 95% CI=0.19-0.72 ; P=0.032, OR=0.339, 95% CI=0.13-0.90 and P=0.006, OR=0.098, 95% CI=0.01-0.74, respectively). Moreover, when EN patient’s HLA haplotypes were compared to controls, two haplotypes were present with higher frequency within EN patient group, HLA- A*02:01~B*27:02~DRB1*16:01 and HLA- A*26:01~B* 38:01~DRB1*04:02 (P=0.002, OR=0 and P=0.054, OR=4.702, 95% CI=0.90-24.45, respectively) while HLA- A*02:01~B*57:01~DRB1*16:01 (P=0.031, OR=0) haplo- type showed a significantly lower frequency. HLA- A*02:01~B*27:05~DRB1*01:01 and HLA-A*01:01~B* 57:01~DRB1*07:01 haplotypes were also less frequent among EN patients but this difference did not reach statistical significance. In conclusion, our results point toward genetic susceptibility of EN and observed differences in both susceptible/protective HLA profiles indicate the necessity of further studies.

Chimerism status evaluation is a routine test performed in post-HSCT period. Aim of the study was to evaluate a qPCR method (GenDx, Utrecht, the Netherlands) applicability for this purpose. Study included 74 recipient/donor pairs tested for informative markers: median of four and six informative markers was found for patients (related and unrelated donor, respectively). Higher sensitivity of qPCR method was confirmed by analysis of recipient post-HSCT samples (N = 800) among which microchimerism (0.1-1% recipient DNA) was detected in 21.8% of cases. The ability to detect less than 1% of minor population, as opposed to the Short Tandem Repeat (STR) method for which 1% is the limit, translated into earlier identification of a disease relapse for four patients in our study sample.

The duration and success rate of an unrelated donor search in hematopoietic stem cell transplantation greatly depends on patients’ HLA typing. Studies about HLA haplotype frequency in Croatian Bone Marrow Registry revealed 50 most frequent HLA-A~B~DRB1 haplotypes (>0.1%). The rest of them were defined as less frequent (

One of the many factors influencing hematopoietic stem cell transplantation (HSCT) is the presence of donor- derived alloreactive NK cells that are formed mainly as the result of KIR-HLA ligand mismatching. Three models for predicting alloreactive NK cell formation exist: the ligand- ligand model, KIR receptor-ligand model and KIR haplotype model. The purpose of this study was to analyze the association of patient and donor KIR ligands, KIR genes and genotypes on HSCT outcome and to see which of the three models the best algorithm to predict HSCT outcomes is. The investigated group consisted of 56 patient-donor pairs that had undergone unrelated HSCT in University Hospital Centre Zagreb. Analysis according to the ligandligand model in the studied group resulted in only four mismatches as opposed to the KIR-ligand model where 31 patient-donor pairs were KIR-ligand mismatched, leading to the conclusion that determining KIR ligand mismatches only following patient-donor HLA typing, without KIR genotyping of donors, is not good prognostic algorithm. The inhibitory KIR-HLA ligand matches/mismatches determined with the KIR-ligand model did not show any beneficial effect of KIR ligand mismatches on any investigated HSCT endpoint. Assigning the AA or Bx KIR genotype to each patient and donor (patient/donor combinations: AA/AA, AA/Bx, Bx/AA and Bx/Bx) enabled its analysis according to the third model and, although no statistically significant difference was found, a trend towards better survival was observed in AA patients receiving a Bx graft. The conclusion is that any of existing models to predict alloreactive NK cell formation and their effect on HSCT outcomes can't give the answer alone and to clarify the KIR influence on HSCT outcome, different approaches in the analyses which encompasses all properties of KIR genes/receptors, KIR-ligands and NK cells together needs to be taken into consideration.

Introductions: Endemic nephropathy (EN) is a chronic tubulointerstitial renal disease associated with the development of cancer of the upper urinary tract (UUC). The Human Leukocyte Antigen (HLA) plays role in immune response and its genes are involved in aetiology of autoimmune diseases as well as in carcinomas. The aim of this study was to investigate the differences between EN patients with and without UUC and HLA-DRB1 genes. Materials and methods: The study included 85 patients aged 58-88 years, with EN, treated at the Urology Department and Dialysis Department, General Hospital “Dr.Josip Benčević” Slavonski Brod, in the period from 2005 to 2018, and 150 healthy matched controls. Peripheral blood of all patients and controls was collected for HLA typing. All individuals were tested for HLA-DRB1 genes using Polymerase Chain Reaction – Sequence Specific Oligos (Luminex technology). Results: Thirteen different HLA-DRB1 genes were observed among EN patients as well as among controls. The most frequent HLA-DRB1 gene in patient’s group was HLA-DRB1*11 (20.7%), while among controls two HLA- DRB1* genes (DRB1*11 and DRB1*16 were observed with the same frequency (15.9%, each). Comparison between tested groups revealed significant P value for DRB1*16 which was significantly more present among patients in comparison to controls (15.9% vs. 8.3% ; P=0.0144). Conclusion: Our preliminary data demonstrated existence of positive association between DRB1*16 gene and EN in our population. The obtained data needs to be confirmed in future studies which should also investigate the distribution of HLA-DRB1 genes among patients with and without UUC, and include other HLA genes.

The association of celiac disease (CD) and HLA class II genes is one of the best established and most documented disease associations of HLA system. The aim of our retrospective study was to determine whether there is a difference in HLA class II allele distribution among individuals who have a higher risk of developing CD (family members of confirmed CD patients or individuals with unconfirmed diagnosis who were referred to our Tissue Typing Centre for HLA typing as a part of the diagnostic procedure) and healthy controls with no history of CD in their family background. For that purpose, we analyzed the results of HLADQ typing performed using the PCR-SSP high resolution method (Olerup GmbH, Vienna, Austria) carried out for patients with CD (N=118), family members of CD patients (N=206), subjects with unconfirmed CD (N=1685) and healthy controls (HC) (N=985). Results show that genotype DQ2.5 cis in both single or double dose was statistically more frequent among all the groups when compared with HC (P

In this paper, we present the case of an HLA-DP immunized patient in the process of kidney re-transplantation for which potential pre-transplantation HLA-DP donor-specific antibodies were detected. For that reason, for the first time in our laboratory practice, prospective cadaveric donor HLA-DP typing was performed during the process of kidney allocation. This case, together with other international study reports and protocols of some transplantation programs, points to the importance of introducing pre-transplant HLA-DP typing in cadaveric kidney transplantation in Croatia.

Human leukocyte antigen (HLA) typing of more than 50, 000 registered volunteer hematopoietic stem cell donors from the Croatian Bone Marrow Donor Registry (CBMDR) provided a large dataset for studying human leukocyte HLA allele polymorphism in the Croatian population which in a certain number of cases lead to the identification of new, rare and very rare HLA alleles. During the last two years we have identified two new HLA alleles and a number of very rare or rare HLA alleles by a sequence-based typing (SBT) method using Olerup SBT kits and Assign™ SBT software for the analysis. HLA-A*01:200 was the first new allele reported in 2016. In the meantime it was confirmed in two siblings, volunteer donors from CBMDR originating from a different part of Croatia (north-west) to the donor in which it was identified for the first time (middle Dalmatia). Further analysis revealed that all three individuals carry the haplotype: HLA-A*01:200~B*08:01~C*07:01~DRB1*03:01~DQB1* 02:01~DPB1*01:01, suggesting the diversity of AH 8.1 haplotype in the Croatian population. The second new allele has been officially assigned by the WHO Nomenclature Committee in December 2017 and the given sequence name is HLA-B*18:37:02. To analyze which of the HLA alleles determined in our laboratory are frequent, rare or very rare, we used the Rare Allele Detector tool incorporated to the Allele Frequency Net Database (AFND) website. Apart from the two new alleles found, three more alleles were determined as very rare: HLA-B*08:78, HLA-B*18:103 and HLA-DRB1*11:04:06. The HLA-A*24:41, HLA-B*07:02: 28, HLA-B*35:03:03, HLA-B*35:101:01, HLA-B*39:40: 01N, HLA-B*44:16, HLA-B*51:78, HLA-DRB1*01:31 and HLA-DRB1*13:23:01 were classified as rare alleles. All this data complements the knowledge about HLA polymorphisms of the Croatian population, provide foundations for further studies in population genetics and highlights once again the importance of recruiting donors from different populations.

Recent studies about HLA polymorphisms in Croatians included HLA haplotype frequency analysis in the Croatian Bone Marrow Registry revealing 50 most common HLAA~ B~DRB1 haplotypes as frequent (>0.1%). The rest of the haplotypes were defined as less frequent (

The aim of the study was to investigate the distribution of HLA-B*44 alleles and determine the common HLA-B*44~C haplotypes in the Croatian population. The subjects included in this study (N=316) were randomly chosen B*44-positive healthy individuals previously typed for the HLA-A, -B, -C and -DRB1 loci. The high resolution level typing of HLA-B and -C loci was performed using the Polymerase Chain Reaction – Sequence Specific Primers (PCR-SSP) method. Among five detected HLA- B*44 alleles the most frequent one was HLA-B*44:02 (41.40%) followed by -B*44:03 (25.70%) and - B*44:27 (18.50%). The HLA-B*44:06 allele was detected only once (0.30%). The analysis of the HLA-C alleles’ distribution showed a predominance of a single HLA-C allele in all four subgroups: HLA-C*05:01 for the HLA-B*44:02-positive individuals, HLA-C*04:01 among HLA-B*44:03- positive subjects, HLA-C*02:02 in the HLA-B*44:05- positive group and HLA-C*07:04 among individuals carrying HLA-B*44:27. The potential application of these data can be found in pre-transplant management of HLA-B*44-positive patients in both hematopoietic stem cell and solid organ transplantation program.

Chimerism analysis is a routine test performed in order to monitor engraftment or relapse after HSCT. A gold- standard method for this test was STR analysis. However, emergence of more sensitive qPCR-based methods has made them a more preferable method for chimerism analysis. The aim of the study was to evaluate applicability of a qPCR based chimerism monitoring method. For that purpose, 74 recipient/donor pairs have been tested using KMRtype Core kit (GenDx, Utrecht, the Netherlands). qPCR was carried out using Applied Biosystems 7500 Real-Time PCR System. Analysis was performed using KMRengine Chimerism Analysis Software. Among tested subjects, 36 patients were transplanted from a related donor, while 38 patients had an unrelated donor. Median number of informative markers (M) was calculated for patients and donors in both groups and was as follows: for patients with related donors M=4 (range 1-7) ; for related donors M=3 (range 0-7) ; for patients with unrelated donors M=6 (range 2-10) ; for unrelated donors M=6 (range 3-12). Analysis of informativeness for individual markers revealed that out of 30 tested markers, eight markers were informative in 15 or more cases for either patient or donor. However, only three markers (KMR013, KMR053, and KMR054) were placed among those eight for both patients and donors. Conversely, several markers showed a low level of informativeness as well as a high percentage (≈20%) of atypical results (KMR011, KMR035, KMR041, KMR057). In conclusion, the qPCR method has been shown to be adequate for majority of our patients. Additional testing was needed for only four patients with a related donor for whom a single informative marker was identified in the first analysis. Improvement of qPCR method applicability in comparison to the initial qPCR-based methods is encouraging and, although the level of PCR-STR method applicability has still not been reached, results of our study justifies implementation of qPCR in routine chimerism monitoring procedure.

Glutenska enteropatija (GE) je bolest kod koje je utvrđena jedna od najjačih povezanosti sa genima sustava HLA. Približno 95% bolesnika s GE su nosioci HLA-DQ2.5 (HLA-DQA1*05-DQB1*02 u cis ili trans konfiguraciji ovisno o tome da li se HLA-DQA1*05 i -DQB1*02 nalaze na istom ili različitim kromosomima) i/ili HLA-DQ8 (HLA-DQA1*03-DQB1*03:02) heterodimera. Cilj rada bio je istražiti raspodjelu alela HLA-DQA1 i -DQB1 kod bolesnika (N=118), osoba s s povećanim rizikom za razvoj GE (članovi obitelji bolesnika (N=206) ili osobe s nepotvrđenom GE (N=1685)) i zdravih osoba (ZK) (N=985). U tu svrhu je za sve ispitanike provedena tipizacija gena HLA-DQA1 i -DQB1 metodom PCR-SSP visoke rezolucije. Rezultati su pokazali da je genotip DQ2.5 cis značajno učestaliji među svim ispitivanim skupinama u odnosu na ZK (P

The Croatian Bone Marrow Donor Registry (CBMDR) with more than 50, 000 registered volunteer hematopoietic stem cell donors provides a large dataset for studying HLA allele polymorphism in the Croatian population which in a certain number of cases leads to the identification of new, rare and very rare HLA alleles. During the last two years we have identified two new HLA alleles and a number of very rare or rare HLA alleles by sequence based typing (SBT) method using Olerup SBT kits and Assign SBT software for the analysis. The first new allele reported in 2016 was HLA-A*01:200 which differs from HLA-A*01:12 in four nucleotide transitions in exon 3 leading to three- amino acid substitution in codons 65, 75 and 76. The allele was confirmed in two siblings, volunteer donors from CBMDR originating from different part of Croatia (north- west) than the donor in which it was identified for the first time (middle Dalmatia) and analysis revealed that all three individuals carry the haplotype: HLA- A*01:200~B*08:01~C*07:01~ DRB1*03:01 ~DQB1*02:01~DPB1*01:01. The second new allele has been officially assigned by the WHO Nomenclature Committee in December 2017 and the given sequence name HLA-B*18:37:02. The new allele differs from HLA-B*18:37 in one change in exon 2 at nt 161 resulting in a non-coding change in codon 53. Applying the Rare Allele Detector tool incorporated to the Allele Frequency Net Database (AFND) website, four alleles found were determined as very rare: HLA-B*08:78, HLA- B*18:103, HLA-DRB1*11:04:06 and HLA-DRB1*12:39. The HLA-A*24:41, HLA-B*07:02:28, HLA-B*35:03:03, HLAB* 35:101:01, HLA-B*39:40:01N, HLA-B*44:16, HLA-B*51:78, HLA-DRB1*01:31, HLA-DRB1*13:23:01, HLA-DRB1*14:111 and HLA-DRB1*16:01:08 were classified as rare alleles. All this data complements the knowledge about the HLA polymorphisms of the Croatian population, provides a foundation for further studies in population genetics and highlights once again the importance of recruiting donors from different populations.